Identification and Subtyping of Avian Influenza Viruses by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Agarose Gel Electrophoresis
DOI:
https://doi.org/10.3923/ijps.2009.465.469Keywords:
Avian influenza, H5N1, India, RT-PCRAbstract
Avian Influenza (AI) is caused by type A influenza virus belonging to the family orthomyxoviridae, which is classified into 16 HA and 9 NA subtypes based on two surface glycoprotein’s haemagglutinin (HA) and neuraminidase (NA). In the present study we did identification and HA-subtyping of avian influenza virus by reverse transcription-PCR (RT-PCR) during the first outbreaks of AI in India during 2006. The avian influenza virus is identified by RT-PCR using a set of primers specific to the nucleoprotein (NP) gene of avian influenza virus. The HA-subtypes of avian influenza virus were determined by running with HA subtype specific primers for H5, H7 and H9 RT-PCR reactions, each using a set of primers specific to one HA-subtype. A total of 10,236 tissue / cloacal swab samples, received at the HSADL from various parts of the country, were processed for isolation of AI virus in embryonated chicken eggs. Out of these, 9 samples originating from poultry in Maharashtra (Navapur and Jalgaon) and Gujarat (Surat) states of India were found positive for H5 virus by RT-PCR. All samples received from outbreaks areas were tested by using all tree subtype specific primers(H5, H7 and H9) only H5 RT-PCR reactions was give the product of expected size, and thus the HA-subtype of the virus is determined. One sample gave the positive result with H9 subtype specific primers. The RT-PCR procedure is rapid and sensitive, and could be used for the identification and HA-subtyping of avian influenza virus in organ homogenates.
References
Altmuller, A., l.M. Kuner, K. Muller, C. Scholtissek and W.M. Fitch, 1991. Genetic relatedness of the nuclioprotien of recent swine, Turkey and human influenza A virus (H1N1) isolates. Virus Res., 22: 79-87.
Atmar, R.L., B.D. Baxter, E.A. Dominguez and L.H. Taber, 1996. Comparison of reverse transcription RT-PCR with tissue culture and other rapid diagnostic assay for detection of type a influenza virus. J. Clin. Microbiol., 34: 2604-2606.
Cherian, T., L. Bobo, M.C. Steinhoff, R.A. Karron and R.H. Yolken, 1994. Use of PCR enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus. J. Clin. Microbiol., 32: 623-628.
Ellis, J.S. and M.C. Zambon, 2002. Molecular diagnosis of influenza. Rev. Med. Virol., 12: 375-389.
Garcia, A., H. Johnson, S.D. Kumar and R.G. Webster, 1998. Efficiency of inactivated H5N2 influenza vaccines against lethal influenza infections. Avian Dis., 42: 248-256.
Hoffmann, E., J. Stech, Y. Guan, R.G. Webster and D.R. Perez, 2001. Universal primer set for the full-length amplification of all influenza a viruses. Arch. Virol., 146: 2275-2289.
Lee, M.S., P.C. Chang, J.H. Shien, M.C. Cheng and S.K. Shieh, 2001. Identification and subtyping of avian influenza viruses by reverse transcription PCR. J. Virol. Methods, 97: 13-22.
Poddar, S.K., 2002. Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis. J. Virol. Methods, 99: 63-70.
Senne, D.A., B. Panigrahy, Y. Kawaoka, J.E. Pearson and J. Suss et al., 1996. Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: Amino acid sequence at the HA cleavage site as a marker of pathogenicity potential. Avian Dis., 40: 425-437.
Stockton, J., J.S. Ellis, M. Saville, J.P. Clewley and M.C. Zambon, 1998. Multiplex RT-PCR for typing and subtyping influenza respiratory syncytial viruses. J. Clin. Microbiol., 36: 2990-2995.
Taubenberger, J.K. and S.P. Layne, 2001. Diagnosis of influenza virus: Coming to grips with the molecular era. Mol. Diagn., 6: 291-305.
WHO., 2002. WHO Manual on Animal Influenza Diagnosis and Surveillance. World Health Organization, Geneva.
Wright, K.E., G.A. Wilson, D. Novosad, C. Dimock, D. Tan and J.M. Weber, 1995. Typing and subtyping of influenza viruses in clinical samples by RT-PCR. J. Clin. Microbiol., 33: 1180-1184.
Wood, G.W., G. Parsons and D.J. Alexander, 1995. Replication of influenza A viruses of high and low pathogenicity for chickens at different sites in chickens and ducks following intra nasal inoculation. Avian Pathol., 24: 245-551.
Yuen, K.Y., P.K.S. Chan, M. Peiris, D.N.C. Tsang and T.L. Que et al., 1998. Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet, 351: 467-471.
Zhang, W.D and D.H. Evans, 1991. Detection and identification of human influenza viruses by polymerase chain reaction. J. Virol. Methods, 33: 165-189.
Downloads
Published
Issue
Section
License
Copyright (c) 2009 Asian Network for Scientific Information
This work is licensed under a Creative Commons Attribution 4.0 International License.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
